Regulatory effect of calcineurin inhibitor, tacrolimus, on IL-6/sIL-6R-mediated RANKL expression through JAK2-STAT3-SOCS3 signaling pathway in fibroblast-like synoviocytes

Abstract

Introduction: This study investigated whether the calcineurin inhibitor, tacrolimus, suppresses receptor activator of NF-κB ligand (RANKL) expression in fibroblast-like synoviocytes (FLS) through regulation of IL-6/Janus activated kinase (JAK2)/signal transducer and activator of transcription-3 (STAT3) and suppressor of cytokine signaling (SOCS3) signaling.

Methods: The expression of RANKL, JAK2, STAT3, and SOCS3 proteins was assessed by western blot analysis, real-time PCR and ELISA in IL-6 combined with soluble IL-6 receptor (sIL-6R)-stimulated rheumatoid arthritis (RA)-FLS with or without tacrolimus treatment. The effects of tacrolimus on synovial inflammation and bone erosion were assessed using mice with arthritis induced by K/BxN serum. Immunofluorescent staining was performed to identify the effect of tacrolimus on RANKL and SOCS3. The tartrate-resistant acid phosphatase staining assay was performed to assess the effect of tacrolimus on osteoclast differentiation.

Results: We found that RANKL expression in RA FLS is regulated by the IL-6/sIL-6R/JAK2/STAT3/SOCS3 pathway. Inhibitory effects of tacrolimus on RANKL expression in a serum-induced arthritis mice model were identified. Tacrolimus inhibits RANKL expression in IL-6/sIL-6R-stimulated FLS by suppressing STAT3. Among negative regulators of the JAK/STAT pathway, such as CIS1, SOCS1, and SOCS3, only SOCS3 is significantly induced by tacrolimus. As compared to dexamethasone and methotrexate, tacrolimus more potently suppresses RANKL expression in FLS. By up-regulating SOCS3, tacrolimus down-regulates activation of the JAK-STAT pathway by IL-6/sIL-6R trans-signaling, thus decreasing RANKL expression in FLS.

Conclusions: These data suggest that tacrolimus might affect the RANKL expression in IL-6 stimulated FLS through STAT3 suppression, together with up-regulation of SOCS3.

Figure 1

The effect of the IL-6/sIL-6R complex on RANKL, JAK2, STAT3 in fibroblast-like synoviocytes (FLS). (A) Stimulation of FLS with IL-6 and sIL-6R at several different concentrations (0, 30, 50, 100 ng of both) for 30 minutes induced RANKL protein expression in a dose-dependent manner. In contrast, expression of OPG protein was gradually inhibited under the same conditions. (B) Treatment with IL-6/sIL-6R (100 ng of both) for 30 minutes in control synoviocytes increased the expression of SOCS3, p-JAK2, p-STAT3, and RANKL proteins. In SOCS3-siRNA transfected FLS, expression of p-JAK2, p-STAT3, and RANKL under IL-6/sIL-6R stimulation (100 ng/ml each) for 30 minutes was significantly increased compared to control synoviocytes. Data are determined in three independent experiments. IL-6, interleukin-6; JAK2, Janus activated kinase; OPG, osteoprotegerin; RANKL, receptor activator of NF-κB ligand; sIL-6R, soluble interleukin-6 receptor; SOCS3, suppressor of cytokine signaling 3; STAT3, signal transducer and activator of transcription-3.

Figure 2

The therapeutic effect of tacrolimus on inflammation and bone erosion in a serum-induced arthritis mouse model. (A) Before immunization of C57BL/6 mice with K/BxN serum, tacrolimus (1 mg/kg) was intraperitoneally introduced four times for a week. At day 10, the experimental mice were sacrificed. Serum-induced arthritic mice pretreated with tacrolimus showed less paw swelling compared to those not treated with tacrolimus. Histological findings demonstrated protection against joint damage in the cartilage, bone, and synovium of tacrolimus-treated mouse joints. (B) Ankle thickness in the tacrolimus-treated mice was significantly less than that of non-treated serum-induced arthritis mice on days 8 and 10 (^*P <0.05). (C) Semi-quantitative analysis for inflammation and bone erosion indicated fewer inflammatory changes and less bone destruction in tacrolimus-treated mice compared to untreated mice (^*P <0.05). (D) Tacrolimus treatment significantly reduced RANKL mRNA expression in the affected joints of mice with serum-induced arthritis (^*P <0.05). In contrast, the reduction in OPG mRNA expression in serum-induced arthritis was reversed with treatment by tacrolimus (^*P <0.05). SOCS3 mRNA expression also was increased in arthritic joints treated with tacrolimus (^*P <0.05 versus serum-induced arthritis not treated with tacrolimus). OPG, osteoprotegerin; RANKL, receptor activator of NF-κB ligand; SIA, serum-induced arthritis; SOCS3, suppressor of cytokine signaling 3; Tac, tacrolimus.

Figure 3

Regulatory effect of tacrolimus on RANKL and OPG expression in FLS under IL-6/sIL-6R stimulation. (A) After pretreatment of cultured FLS with IL-6/sIL-6R (100 ng of both), incubation with tacrolimus at several concentrations led to a consistently marked reduction of RANKL at the mRNA (^*P <0.001 at 10, 100, 1,000 nM of tacrolimus) and protein levels (^‡P <0.05 at 100 nM and ^†P <0.01 1,000 nM of tacrolimus). In contrast, tacrolimus at dosages of 100 and 1,000 nM significantly increased levels of OPG mRNA (^*P <0.001 for each dosage). Expression of OPG protein was consistently observed (^†P <0.01 of 100 and 1,000 nM). (B) In western blot analysis for RANKL expression after tacrolimus treatment, tacrolimus was found to inhibit RANKL expression in FLS stimulated by IL-6/sIL-6R (100 ng of both). In contrast, OPG expression was increased following tacrolimus treatment. (C) Tacrolimus was shown to inhibit the expression of RANKL in FLS after stimulation with IL-6/sIL-6R in the immunofluorescence assay. (D) The inhibitory effect of tacrolimus (1 µM) on RANKL expression was more prominent than that of other anti-inflammatory drugs such as methotrexate (1 µg) and dexamethasone (1 µg). OPG mRNA expression was increased by tacrolimus and methotrexate but not by dexamethasone. (^*P <0.001, ^†P <0.01, ^‡P <0.05 versus IL-6/sIL-6R-treated FLS). Data are determined in three independent experiments. FLS, fibroblast-like synociocytes; IL-6, interleukin-6; OPG, osteoprotegerin; RANKL, receptor activator of NF-κB ligand; sIL-6R, soluble interleukin-6 receptor.

Figure 4

The effect of tacrolimus on JAK2, STAT3, and SOCS3 in IL-6/sIL-6R-stimulated FLS. (A) Stimulation with IL-6/sIL-6R (100 ng of both) induced phosphorylation of JAK2 and STAT3. However, tacrolimus reversed these changes, thereby significantly reducing the expression of p-JAK2 and p-STAT3. (B) Tacrolimus treatment of IL-6/sIL-6R-stimulated FLS potently suppressed IL-6 expression. Among negative regulators of the JAK-STAT signaling pathway, prominent induction of SOCS3 mRNA expression was induced by tacrolimus (^†P <0.05 at 100 and 1,000 nM) in comparison to IL-6/sIL-6R-stimulated FLS. Expression of SOCS1 and CIS1 mRNA was not similarly induced. (C) Tacrolimus enhanced the level of SOCS3 protein in IL-6/sIL-6R-treated FLS in a dose-dependent manner. (D) In SOCS3 knockdown FLS, IL-6/sIL-6R induced overexpression of RANKL, p-NF-κB and NFATc1. In contrast, addition of tacrolimus induced SOCS3 expression and attenuated RANKL expression. The protein levels of p-NF-κB and NFATc1 were significantly reduced, in comparison to those in SOCS3 knockdown FLS without tacrolimus. (E) The immunofluorescence assay indicated the presence of an increased number of SOCS3-positive cells after treatment with tacrolimus compared to controls. Data are determined in three independent experiments. CIS1, cytokine-inducible SH2; IL-6, interleukin-6; JAK2, Janus activated kinase; RANKL, receptor activator of NF-κB ligand; sIL-6R, soluble interleukin-6 receptor; SOCS1, suppressor of cytokine signaling 1; SOCS3, suppressor of cytokine signaling 3; STAT3, signal transducer and activator of transcription 3.

Figure 5

The effect of tacrolimus on the formation of TRAP(+) multinucleated cells. (A) TRAP staining assay showed that 60 ng/ml of RANKL and 50 ng/ml of M-CSF induced differentiation of PBMCs into TRAP (+) multinucleated cells, implicating osteoclasts (a). However, addition of tacrolimus gradually decreased the number of TRAP (+) multinucleated osteoclasts in a tacrolimus dose-dependent manner (b to d) (magnitude × 200). (B) Tacrolimus significantly suppressed number of TRAP(+) cells at 0.5 Mm and 1.0 Mm doses (^*P <0.05 and ^†P <0.01). M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of NF-κB ligand; TRAP, tartrate-resistant acid phosphatase.

Figure 6

Summary for the effect of calcineurin inhibitor, tacrolimus, on the regulation of RANKL expression through IL-6/sIL-6R/JAK2/STAT3/SOCS3 pathway. IL-6, interleukin-6; JAK2, Janus activated kinase; RANKL, receptor activator of NF-κB ligand; sIL-6R, soluble interleukin-6 receptor; SOCS3, suppressor of cytokine signaling 3; STAT3, signal transducer and activator of transcription 3.